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標題: 楊桃細菌性斑點病菌保留因子區內基因之特性分析
Characterization of effector genes in conserved effector locus of Pseudomonas syringae pv. averrhoi
作者: 許嘉真
Hsu, Chia-Chen
關鍵字: Pseudomonas syringae pv. averrhoi;楊桃細菌性斑點病菌;type III secretion system (T3SS);conserved effector locus (CEL);第三型分泌系統;保留性因子區
出版社: 生物科技學研究所
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楊桃細菌性斑點病是由Pseudomonas syringae pv. averrhoi (Pav)所引起的病害。此菌為革蘭氏陰性菌,桿狀,在楊桃植株上產生的病徵為葉片上有紫紅色斑點,周圍有明顯黃暈現象,且葉片易黃化脫落。其分子致病機制主要受控於病原島嶼 (Pathogenicity island, Pai)基因序列所轉譯的蛋白質,Pai為一個三組基因群組成的鑲嵌結構,其中hrp/hrc基因組是在不同病原型中具高度保留性的核心區域 (core region),主要形成第三型分泌系統,此分泌系統可將病原菌的effector分泌至寄主細胞內,而在兩側分別為可交換因子區 (exchangeable effector locus, EEL)與保留因子區 (conserved effector locus, CEL)。由楊桃細菌性斑點病菌Pav HL1菌株中選殖到16.7 kb的保留因子區,此區域基因排列組成及序列與P. s. pv. phaseolicola (Pph) 1448A最為相近,其中orf1、avrE、avrF、orf6基因產物為已知或可能是有效蛋白質,而hrpW基因與hopM1、hopAA1基因分別由於產生突變或鹼基缺失導致基因可能無法表現。利用pK18mobsacB為載體及同源重組作用,構築orf1、avrE、avrF及orf6等基因突變株,發現orf1基因缺失會降低Pav引發過敏性反應的能力,顯示Orf1蛋白與病原菌引發過敏性反應有關,而AvrE與AvrF蛋白則與過敏性反應無關;在互補試驗中,發現在質體上表現的Orf6蛋白能抑制過敏性反應的產生。分析各基因突變菌株在寄主楊桃上的生長能力,發現在接種九天後,orf1基因缺失菌株菌量為野生菌株菌量之1/50;avrE或avrF基因缺失菌株菌量均為野生菌株菌量之1/100,但avrF基因缺失於初期菌量的影響較為輕微;而orf6基因缺失菌株降低的菌量與野生菌株無顯著差異。ORF1具有transglycosylase motif,能編碼出肽聚醣水解酶,目前推測其功能應造成病原菌細胞壁鬆散而能在其上構築第三型分泌系統的針狀構造。研究中構築一orf1-FLAG基因,並利用M2單株抗體偵測其所表現的融合蛋白,以探討Orf1蛋白在細胞中的位置,發現Orf1蛋白似乎未被分泌至細胞外。將Orf6表現於帶有Psy 61 hrp/hrc基因組 (pHIR11)的Escherichia coli MC4100中,發現Orf6蛋白可能會經由第三型蛋白分泌系統被分泌至菌體胞外,並能抑制由HopA1所引起的過敏性反應。

Bacterial spot of carambola is caused by Pseudomonas syringae pv. averrhoi (Pav), which is a gram-negative, rod-shaped bacterium. The typical symptoms on leaves are purple spots surrounded by yellow haloes, and the infected leaves turn yellow and fall easily. Pav possesses a Hrp Pathogenicity island (Pai) responsible for pathogenicity on its host and hypersensitive response (HR) on resistant plants. The Hrp Pai consists of three regions: EEL (exchangeable effector locus), hrp/hrc cluster (also called core region) which encodes a type III secretion system (T3SS) that facilitates the effectors to be translocated into host cell, and CEL (conserved effector locus). A 16.7 kb DNA fragment containing CEL was cloned from Pav strain HL1. The DNA sequences of CEL show the highest similarity to the corresponding region of P. s. pv. phaseolicola (Pph) 1448A. Among nine orfs contained in this CEL, orf1, avrE, avrF and orf6 are predicted to be putative effector genes, hrpW carries a nonsense mutation and probably encodes only N-terminal amino acids which possesses the harpin domain, hopM and hopAA1 are both truncated. The mutant strains of orf1, avrE and orf6 were constructed by means of homologous recombination using pK18mobsacB as a vector. Based on the HR elicitation on nonhost tobacco leaves, orf1 mutant elicited the delayed HR, suggesting that Orf1 might be involved in the elicitation of HR. A complementation assay verified that the expression of native orf6 gene in trans can suppress the HR elicitation in nonhost induced by Pav HL1, but AvrE and AvrF are not related to the elicitation of HR. All mutants tested here reduced their ability to grow in host carambola, thus all of them contribute to the proliferation and accumulation in host plant. Orf1 is a transglycosylase-like protein and may have peptidoglycan hydrosylase activity which causes cell leakage and facilitates the assembly of the T3SS. In order to elucidate the cellular location of Orf1, a FLAG tag which can be easily detected by M2 monoclonal antibody in immunoblotting was fused with orf1. A western blot analysis revealed that Orf1-FLAG fusion protein is mainly located in the total cell pellet fraction. By using an E. coli MC4100 (pHIR11) system, it was revealed that Orf6 may act inside plant cells to block the HR elicitation in tobacco by the pHIR11-encoded effector HopA1.
其他識別: U0005-2606200712240200
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